Summary Oxidative DNA damage is continuously generated in cells by both endogenous and exogenous genotoxicants. The damage leads to mutation and cell death and is the cause of cellular transformation and eventual carcinogenesis. DNA base excision repair (BER) is the cell[unreadable]s primary defense mechanism to prevent such mutations. The BER enzyme at the center of activity is apurinic/apyrimidinic endonuclease 1 (APE1), which reacts with the DNA lesions to generate 3[unreadable]-OH termini that are then processed by the DNA synthesis machineries. Our recent studies indicate that APE1 is essential for cellular viability. Paradoxically, however, APE1 (and BER) may become harmful to the body, since cancer cells can acquire drug- and radio-resistance due to increased APE1 expression, giving the cells a better chance of survival. Therefore, changes in APE1[unreadable]s enzymatic properties are crucial not only to cells but also to a whole body. We have found that APE1 is ubiquitinated by the mouse double minute 2 protein, Mdm2, and regulated by p53 upon DNA damage generation. The ubiquitination occurs at specific Lys residues in the APE1 polypeptide. It appears to have significant effects on the functions of APE1 and other cellular factors including XRCC1 and p53 that interact directly with APE1. The central hypothesis of this project is that APE1 ubiquitination, regulated by p53/Mdm2, modulates the cellular APE1 level and activity during the DNA damage response and apoptosis. The Specific Aims of this project are: (1) to determine the effect of APE1 ubiquitination on its functions, (2) to examine the interaction of ubiquitinated APE1 with other BER enzymes and p53/Mdm2, and (3) to determine the regulatory pathways through which APE1 ubiquitination occurs in response to DNA damage. We will use biochemical and cell biological techniques as the primary means of identifying the specific ubiquitination mechanism that regulates APE1 in vivo. Understanding the ubiquitination of APE1 will significantly increase our knowledge of the cellular defense mechanism against carcinogenesis.